Carboxylic acids are important high volume commodities in the chemical industry. For example, it is estimated that the 1982 worldwide capacity for adipic acid is about five billion pounds.
Adipic acid is produced by oxidation of cyclohexane or cyclohexanol with nitric acid in the presence of a vanadium-copper catalyst. Other methods of synthesizing adipic acid include 1,3-butadiene carbonylation with carbon monoxide followed by hydrolysis; methyl acrylate dimerization; and 1,4-butanediol carbonylation.
Recent biotechnical advances have increased interest in the potential application of bioconversion systems for the production of high volume chemicals such as adipic acid and other commercially established commodities.
One prospective new method of synthesizing a carboxylic acid such as adipic acid is by the hydrogenation of muconic acid, which is a diolefinically unsaturated adipic acid derivative: ##STR1##
A potentially convenient source of muconic acid is by the microbiological oxidation of various hydrocarbon substrates. Microbiological oxidation of hydrocarbons is reviewed in Applied Microbiology, 9(5), 383(1961) and in "Advances in Enzymology", 27, 469-546(1965) by Interscience Publishers.
The Journal of Biological Chemistry, 241(16), 3776 (1966) reports the conversion of catechol and protocatechuate to .beta.-ketoadipate by Pseudomonas putida. The conversion of catechol proceeds by the ortho pathway via a muconic acid intermediate: ##STR2## The chemical structures illustrated in the reaction scheme are catechol, muconic acid, muconolactone, .beta.-ketoadipate enollactone and .beta.-ketoadipate, respectively.
In the Journal Of Bacteriology, 134, 756(1978) there is reported a study of the ubiquity of plasmids in coding for toluene and xylene metabolism in soil bacteria. One of the mutant strains of Pseudomonas putida isolated had the ability to metabolize toluene via benzyl alcohol, benzaldehyde, benzoic acid and catechol by the ortho pathway through .beta.-ketoadipate to a biomass and carbon dioxide.
The enzymes functioning in the toluene metabolism by the ortho pathway included toluene mono-oxygenase, benzyl alcohol dehydrogenase, benzaldehyde dehydrogenase, benzoate oxygenase, dihydrodihydroxybenzoate dehydrogenase, catechol 1,2-oxygenase and muconate lactonizing enzyme. The subsequently formed .beta.-ketoadipate was further assimilated to biomass and carbon dioxide. The mutant strains that metabolized toluene via the ortho pathway did not accumulate muconic acid, since the said muconic acid metabolite was further transformed in the presence of muconate lactonizing enzyme.
No known naturally occurring microorganisms (e.g., Pseudomonas putida) are known that metabolize an aromatic hydrocarbon substrate such as toluene by the ortho pathway via muconic acid and .beta.-ketoadipate. Wild strains metabolize aromatic hydrocarbon substrates by the meta pathway via 2-hydroxymuconic semialdehyde instead of a muconic acid intermediate. Catechol 2,3-oxygenase is functional rather than catechol 1,2-oxygenase.
Thus, the potential of microbiological oxidation of toluene as a convenient source of muconic acid requires the construction of mutant strains of microorganisms which (1) metabolize toluene by means of the ortho pathway, and (2) allow the accumulation of muconic acid without further assimilation.
The said construction of the desirable mutant strains recently has been achieved, as exemplified by Pseudomonas putida Biotype A strain ATCC No. 31916 and genotypically related mutants.
As a consequence of the prospect of large scale bioconversion systems for production of carboxylic acid type compounds from lower cost hydrocarbon substrates, the problems of fermentation system stability, effective biocatalyst activity and consequential bioconversion product formation and accumulation, and of efficient recovery of extracellular bioconversion products contained in fermentation culture media are of increasing significance. The product inhibition of enzymatic activity by an accumulated carboxylic acid metabolite in a fermentation medium is a serious obstacle to high rate production of the carboxylic acid metabolite as a desired product of the process.
Accordingly, it is an object of this invention to provide a bioconversion process for converting a non-growth aromatic hydrocarbon to an extracellular accumulated quantity of carboxylic acid metabolite with a sustained high level of biocatalytic activity.
It is a further object of this invention to provide a continuous fermentation process for bio-oxidation of toluene via the ortho pathway to accumulated extracellular muconic acid with a sustained high reactor productivity, and to provide for the recovery of the accumulated muconic acid product.
Other objects and advantages of the present invention shall become apparent from the accompanying description and examples.